A first step in many chemical and biological applications is the discretization of sample volumes into small individual volumes for subsequent assays and analysis, where the assay or analysis can be of the same type or different types. The diverse variety of these applications highlights a need for different sample generation techniques with distinct advantages. Towards this end, several methods exist. Traditional methods of generating small volumes of sample include the use of a nebulizer to create an aerosol and the mixing of a sample with an immiscible phase to create an emulsion. However, these methods have limiting properties. The droplets formed by a nebulizer exhibit a large range of sizes. Similarly, mixing two immiscible phases to form an emulsion does not produce monodisperse droplets. Furthermore, the droplets formed by either method are difficult to individually manipulate and analyze once formed. In addition, neither of these methods would be well suited for the continuous monitoring that is necessary for in situ assays.
A common approach to forming a discretized sample is the dispensing of small volumes into small wells or microwells. While this is a successful method of creating an array of spatially localized samples, the individual well filling can be a tedious endeavor. Furthermore, human error can get in the way of dispensing homogeneous volumes. To avoid this, researchers have developed special dispensing equipment, such as robotic pipettes. However, the need for specialized dispensing equipment limits the flexibility of these microwell platforms, as well as raising the cost. In addition, it is difficult to work with very small volumes (below 0.5 μL) because of the relatively high dispensing volumes of pipettes as well as problems with sample evaporation. (Jackman, R. J., Duffy, D. C., Ostuni, E., Willmore, N. D., and Whitesides, G. M. Anal Chem. 1998, 70, 11, 2280-2287, Morrison, T., Hurley, J., Garcia, J.; Yoder, K., Katz, A., Roberts, D., et. al. Nucleic Acids Research 2006 34, 18, 15), incorporated by reference in its entirety herein for all purposes.
Two methods of sample array generation which do not rely on manual sample dispensing are the arrangement of droplets on patterned self assembled monolayers (SAMs) (Biebuyk, H. A., Whitesides, G. M. Langmuir 1994, 10, 2790.), and droplet manipulation using electrowetting (Pollack, M. G., Fair, R. B., Shenderov, R. B. Appl. Phys. Lett. 2000, 77, 1725), incorporated by reference in its entirety herein for all purposes. In both cases, the sample is discretized by the surface properties of a substrate, inducing a more uniform droplet volume. However, electrowetting mostly works for samples that are above the nanoliter scale and require sophisticated patterns of electrodes, greatly limiting its utility. Patterned SAMs can be used to generate smaller volumes, however the preparation of these surfaces can be labor intensive. In addition, surfaces must be coated in a layer of gold, and then derivatized in order to achieve the patterned surface. This has the potential to limit the forms of analysis possible, since the substrate is often opaque.
Recently new methods of sample volume discretization have emerged. These methods include the use of microfluidic valves to separate volumes from each other (Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., and Quake, S. R. Science 2000 288(5463):113-116) or the generation of a droplet stream (Sgro, A., Allen, P. B., Chiu, D. T. Anal. Chem. 2007, 79, 4845-4851.) or individual droplets (Lorenz, R. M., Edgar, J. S., Jeffries, G. D. M., Chiu, D. T. Anal. Chem. 2006, 78, 6433-6439.), incorporated by reference in its entirety herein for all purposes.
These methods, however, may suffer from certain drawbacks. For example, the use of valves and pumps require complex fluidic control and fabricated devices that tend to be expensive, especially if many discrete volumes (hundreds to thousands) are involved. The use of steady state methods to generate a stream of droplets makes the subsequent manipulation and immobilization of droplets difficult, but more importantly, because it is a steady state method, it often involves loss of sample volume, that is, there is some initial waste of the sample because a steady state flow stream must be established first prior to the regular formation of droplets. Additionally, such flow methods require accurate control of flow rates, which also increases the complexity and expense of the final device or instrument.
Accordingly, there is a need in the art for novel approaches for the manipulation of sample volumes.